Some techniques are poor in most cases, some good in many cases and some are good in most cases, but none are reliable in every case
C) STR Method (Fig. 5.4)
• Tandem repeated DNA sequences are present in human genome and they show variability in different individuals.
• These tandemly repeated regions of DNA are classified into several groups depending on the size of the repeat region such as:
1. Minisatellites – variable number of tandem repeats – VNTRs
2. Microsatellites – short tandem repeats – STR – have repeats with 2-5 bp (Fig. 5.5).
• STR is a PCR technique that may replace RFLP. This technique is rapid and can be performed on small quanti-ties of DNA.
• STR is done by:
1. Isolating the DNA
2. Replicating the STR fragments by PCR 3. Performing gel electrophoresis
4. Identifying the fragments using stains or laser tech-nique.
Advantages of StR technique 1. Rapid
2. Small sample required
3. Degraded DNA may be typed using STR.
Advantages of DNA Fingerprinting
1. Conclusive method of identification of an individual 2. Method can be applied to old stains or biological
material
3. Small quantity of sample is required.
Fig. 5.4: STR method of DNA typing
Fig. 5.5: Short tandem repeats
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Forensic Medicine
Disadvantages of DNA Fingerprinting
1. DNA profiling cannot differentiate between monozy-gotic twins
2. Expensive
3. Interpretation requires trained manpower 4. Susceptible for contamination.
appLIcatIon oF dna proFILIng 1. To establish identity of a person in
2. To acquit a falsely implicated person from such similar crime.
• False implication on a person being father of a cer-tain child.
7. Extortion cases.
8. Immigration cases.
9. Determination of twin zygosity.
10. To identify sex.
Factors influencing the Applicability of DNA technique
1. Non-human DNA 2. Degradation of sample 3. Contamination of sample 4. Multiple contributors to sample.
Sources of DNA Contamination8
1.
• Few cells are sufficient to obtain DNA information to help in the investigation.
• DNA evidence (sample) can be obtained from the scene of crime, from clinical examination of person or from dead bodies.
• Common objects or items that can be helpful to obtain DNA material,9 found at scene of crime or during autopsy, are mentioned in Table 5.1.
Table 5.1: Common evidentiary material and sources found at scene of crime Evidence/material Sources of DNA
Weapons Blood, hair, tissue
Bullet Blood, tissue
Clothes Blood, semen, sweat
Toothbrush Saliva
Used cigarette/butts Saliva
Used condom Semen, vaginal cell, penile cell, hairs
Bite mark Saliva
Finger scrapings/content Tissue, blood
coLLectIon, preservatIon and foRWARDiNg of SAMPle
Collection, preservation and forwarding of sample for DNA analysis are equally important. Unless the samples are properly collected and preserved, it will not be useful for investigation. The consequences of improper collection and preservation of samples are:
1. If not properly collected, the biological activity of sample may be lost.
2. If improperly packed, cross contamination may occur.
3. If improperly preserved, decomposition and degra-dation may occur. DNA extraction from degraded sample is difficult and challenging.10
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Collection and preservation of sample
1. Dried blood stains/samples can be lifted from non-porous surface with conventional adhesive tape.
2. Liquid blood: Collect 2–5 ml intravenous blood, place it in clean and sterile test tube. Add four per-cent EDTA as preservative.11
3. Semen/vaginal swabs should be preserved in clean and sterile container. In gang rape case, more than two vaginal samples/swabs should be collected and send in separate tubes. In case of delay, vaginal swabs should be stored12 at 4°C.
4. Whenever swabs from saliva are taken, they should be air-dried. Preferably double swab technique should be used. In this method, initially first wet cotton swab should be taken followed by dry cotton swab.13 5. Clothes should be air dried at and packed in paper,
never use polythene bag or plastic sheet to wrap.
Store at room temperature.
6. In exhumation cases – dry tissues are placed in a sterile container without adding any preservative and sent to laboratory at room temperature.
7. It is possible to use fetal tissue for DNA typing. The optimal sample consists of fetal blood obtained by heart puncture. But this is possible in older fetus.
In young fetus, analysis of chorionic villi may pro-vide the fetal pattern without maternal contamination.
Other tissue suitable for DNA analysis is – quadri-ceps muscle or ribs.14
8. Alternatively, as such fetus can be sent in normal saline or DMSO (dimethyl sulphoxide) saturated with sodium chloride (NaCl). The jar-containing fetus should be placed in ice box.12
9. If fetus is macerated, fetal lungs and brain tissues are more suitable for DNA typing.14
10. The samples and preservatives used are summarized in Table 5.2.
Care to be taken
• Short-term storage (2 weeks) at 25°C – 37°C temperature can cause degradation of DNA extracted from seminal stains. PCR amplification is difficult in such cases.
• While storing and transporting clean paper packets are ideal for trace evidences whereas airtight containers are suitable for soft tissue sample.
• Samples should never be packed in plastic bag (polythene bag) as it retains moisture and moisture may contribute for DNA degradation or at times may help to grow bacte-ria. Bacterial growth may pose difficulty in DNA analysis.
• Blood for DNA should not be collected immediately from person who has received blood transfusion.
The blood can be collected after 4 to 6 month after receiving transfusion.
Table 5.2: Samples and preservatives used
Sample Method for collection/packing Preservative
Blood - Use clean sterile container
- Add recommended preservative - Bring the sample on ice
4% EDTA
Tissue, muscle, skin, organs Place sample in clean sterile container Normal saline or Keep tissue as it is in -20°C
Teeth Air dry, place them in clean and sterile container No preservative Scalp hairs with root Air dry sample, place in clean and sterile container No preservative
Bone Air dry, wrap in clean paper No preservative
Blood stained clothes/ scrapings Air dry sample, pack in clean paper No preservative
Semen stains Air dry clothes and pack in clean paper No preservative
Vaginal swabs Air dry, place the swabs in dry, clean and sterile container No preservative Vaginal smear On glass slide, place slides in clean paper packet No preservative Vaginal fluid/seminal fluid Collect in clean and sterile container Frozen solid15
Saliva Collect in clean and sterile container Frozen solid15
Saliva stain on fabric Air dry and pack in clean paper No preservative
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reFerences
1. Jeffreys AJ, Brookfield JFY, Semenoff R. Positive identifica-tion of an immigraidentifica-tion test case using human DNA finger-prints. Nature 1985; 317: 818 – 9.
2. Fierro MF. Identification of human remains. In: Spitz WU (ed) Spitz & Fisher’s Medicolegal investigation of Death, 3rd edn.
1993. Charlas C Thomas Publisher, USA. 71 – 117.
3. Tirpude BH, Sutay SS, Sheikh N. Crime solving tool – the DNA (a review article). J Mediclegal Assoc Maharashtra 2003;
15: 1 – 7.
4. Bardale R, Dixit PG, Deokar S. Mistaken identity, unfolded:
A case report. Milestone 2005; 4:20–22.
5. Haglund WD, Reay DT, Tepper SL. Identification of decom- posed human remains by deoxyribonucleic acid (DNA) profil-ing. J Forensic Sci 1990; 35: 724 – 9.
6. Raina A, Dogra TD, Murty OP. Identification of embalmed tissues using DNA fingerprinting techniques. In: Ajmani ML (Ed) Embalming, Principles and Legal Aspects, 1st edn. 1998.
Jaypee Brothers Medical Publishers, New Delhi.
7. Helminen P, Ehnholm C, Lokki ML, Jeffrey A, Peltonen L.
Application of DNA fingerprinting to paternity determinations.
Lancet 1988; 12: 574 – 6.
8. Pramanik P, Sharma SK, Murty OP. Effect of DNA sample collection, storage and transport on DNA fingerprinting: an overview. Int J Med Toxicol Legal Med 2006; 8: 11 – 3.
9. Vij K, Biswas R. In: Basis of DNA and evidentiary issues, 1st edn.
2004. Jaypee Brothers Medical Publishers (P) Ltd., New Delhi.
10. Lee HC, Ladd CA, Scherezinger CA, Bourke MT. Forensic application of DNA typing. Part 2: collection preservation of DNA evidence. Am J Forensic Med Pathol 1998; 19: 10 – 18.
11. Manual published by Directorate of Forensic Science Laboratories, Home Dept., State of Maharashtra-Mumbai, 2007.
12. Rao GV. Collection and forwarding of forensic samples for DNA fingerprinting. J Indian Acad Forensic Sci 1997; 36: 69 – 73.
13. Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E.
An improved method to recover saliva from human skin: the double swab technique. J Forensic Sci 1997; 42: 320 – 2.
14. Ludes BP, Mangin PD, Malicier DJ, Chalumeau AN, Chaumont AJ. Parentage determination on aborted fetal material through deoxyribonucleic acid (DNA) profiling. J Forensic Sci 1991;
36: 1219 – 23.
15. Kagne RN, Ambade VN, Pathak AG. DNA fingerprinting:
Collection, preservation and dispatch of biological sample.
Souvenir of Formedicon 2004, XIIth Annual Conference of Medicolegal Association of Maharashtra, 2004. 39 – 42.
Autopsy
Synonyms: Also called as necropsy or postmortem examination Autopsy means (autos = self, opis = view) to see for oneself.
Necropsy (necros = dead, opis = view) is most accurate term for the investigative dissection of the dead body, but the term autopsy is commonly used and is more popular. Postmortem (post = after, mortem = death) examination is an alternative term used but suffers from lack of precision about the extent of examination. In some countries, many bodies are disposed off after external examination without dissection, in such situ
ation; the procedure is called as postmortem examination.1 Types of Autopsy
Autopsy may be (Fig. 6.1):
1. Clinical autopsy (pathological autopsy or academic autopsy)
2. Medicolegal autopsy (forensic autopsy) Clinical autopsy
• It is done by Medical Practitioner or treating doctor with the consent of relatives to know the diagnosis or to con
firm the diagnosis.
• Here the autopsy may be complete or incomplete (partial) depending upon the consent obtained for that part of body.
• It is not done under legal obligation therefore no requisi
tion from police is required.
• For doing clinical autopsy, consent of relatives is must. With
out consent, a doctor cannot proceed for clinical autopsy.